Pyrrolo-indole and pyrrolo-quinoline derivatives as prodrugs for tumour treatment

ABSTRACT

Compounds of the general formula (I) or (IA) in which X is H, Y is a leaving group, R 1  and optionally also R 3  preferably being an aromatic DNA binding subunit are prodrug analogues of duocarmycin. The compounds are expected to be hydroxylated at the carbon atom to which X is joined, by cytochrome P450, in particular by CYP1B1, expressed at high levels in tumors. The prodrug is expected to be activated preferentially in tumor cells, where it will act as a DNA alkylating agent preventing cell division.

The present invention concerns aromatic oxidation/hydroxylationactivated prodrugs, particularly anti-tumour prodrugs and those whichare specifically activated by the oxidation/hydroxylation activities ofthe cytochrome P450 family of enzymes.

Many conventional cytotoxic drugs are known that can be used fortherapeutic purposes. However, they typically suffer from the problemthat they are generally cytotoxic and therefore may affect cells otherthan those that are required to be destroyed. This can be alleviated tosome extent by the use of targeted drug delivery systems, for exampledirect injection to a site of tumourous tissue or, e.g. binding thecytotoxic agent to an antibody that specifically recognises an antigendisplayed only on the cancer cell surface. Alternatively,electromagnetic radiation may be used to cause chemical alteration in anagent at a desired site such that it becomes cytotoxic. However, all ofthese techniques have, to a greater or lesser extent, certainlimitations and disadvantages.

The compound (+)—CC-1065 and the duocarmycins are naturally occurringrepresentatives of a class of DNA alkylating agents. The naturallyoccurring compounds consist of a DNA alkylating unit based upon apyrrolo[3,2-e]indole core, with one or two sub units, conferring DNAbinding capabilities. CC-1065 and duocarmycin A comprise a spirocycliccyclopropane group responsible for the DNA alkylation properties.Duocarmycin B₂, C₂ and D₂ are believed to be precursors for cyclopropaneactives, and comprise a substituted (by a leaving group) methyl group atthe eight position on the dihydro pyrrole ring. CC-1065 has beensynthesised by various routes, summarised by Boger et al. in Chem. Rev.1997, 97, 787-828.

In U.S. Pat. No. 4,413,132 the first synthesis of the left hand sub-unitof CC-1065 was described. The synthesis is based on a Winstein Ar-3′alkylation in which the cyclopropane ring is introduced. In a previousstep, the A ring (of the indole core) is introduced by reaction of ananiline with an α-thiomethylester using chemistry based on Gassman'sOxindole synthesis. The aniline has a protected phenolic hydroxyl grouportho to the NH₂ group, which, in the final product, is believed to becrucial for DNA alkylation. CC-1065 has broad antitumour activity but istoo toxic against normal cells to be clinically useful. Attempts havebeen made to target the delivery of CC-1065 and analogues by conjugatingthe drug via the DNA binding subunit to polymers, or specific bindingagents such as antibodies or biotin described in U.S. Pat. No.5,843,937. Boger et al in Synthesis 1999 SI, 1505-1509 describedprodrugs of 1,2,9,9a-tetrahydrocyclopropa(c)benz[e]indol-4-one, in whichthe cyclopropane ring-opened version of the compounds were derivatisedby reaction of the phenolic group to form esters and carbamates.

In J. Am. Chem. Soc. (1991), 113, 3980-'83 Boger et al describe a studyto identify features of CC-1065 analogues contributing to theselectivity of the DNA-alkylation. The compounds tested in vitro hadalkylating subunits based on 2,3-dihydroindole and included the6-deshydroxy analogues. These were shown to have some DNA alkylatingproperties though at concentrations 10⁴ times higher than that of the6-hydroxy compounds.

Tercel et al, in J. Org. Chem. (1999) 64, 5946-5953 describe aminoanalogues of CC-1065 (i.e. in which the phenolichydroxy of the B-ring isreplaced by amino). These are synthesised by nitrating the benzene ringin a late stage intermediaet having a methylol group attached to thedihydropyrrole ring.

The present invention relates to precursors of CC-1065 and itsanalogues, which do not have the hydroxyl group in the B ring of thealkylating sub unit, and which are hence inactive as DNA alkylatingagents themselves, as well as their synthesis and intermediates usedtherein.

It has been reported (Murray, G. I. et al., 15 Jul. 1997, CancerResearch, 57m 3026-3031 and WO-A-9712246) that the enzyme CYP1B1, amember of the cytochrome P450 (CYP) family of xenobiotic metabolisingenzymes, is expressed at a high frequency in a range of human cancers,including cancers of the breast, colon, lung, oesophagus, skin, lymphnode, brain and testes, and that it is not detectable in normal tissues.This led to the conclusion that the expression of cytochrome P450isoforms in tumour cells provides a molecular target for the developmentof new antitumour drugs that could be selectively activated by the CYPenzymes in tumour cells, although no drug examples were given. A numberof other CYP isoforms have been shown to be expressed in varioustumours. Many of the CYP's expressed in tumours are mentioned inPatterson, L H et al, (1999) Anticancer Drug Des. 14(6), 473-486.

In WO-A-99/40056 prodrugs of styrene- and chalcone-derivatives aredescribed. The respective hydroxylated forms of the prodrugs, formed insitu, are potent tyrosine kinase (TK) inhibitors. Inhibition of TKactivity contributes to tumour inhibition and cell destruction. Theprodrugs were shown to be activated by microsomal preparationsexpressing CYP1B1 enzyme, and to have cytotoxic activity against celllines expressing the same enzyme, whilst having much lower cytotoxicactivity against cell lines not expressing the enzyme.

The present invention is directed to a new class of prodrugs which areexpected to be hydroxylated in situ by CYP enzymes, in particularenzymes expressed at high levels in tumours as described in Patterson LH, et al, op. cit. In particular the prodrugs are believed to bemetabolisable by CYP1B1enzyme. Some of the compounds are new. Thepresent invention relates to the first therapeutic use of a broad rangeof compounds.

There is provided according to the first aspect of the invention the newuse of a compound of the general formula I or a salt thereof in themanufacture of a composition for use in a method of treatment by therapyof an animal:

in which X is H;

Y is a leaving group

R¹ is —Ar, —NH₂, R⁸ or OR⁸;

R² and R⁴ are each independently selected from H, C₁₋₄ alkyl, —OH, C₁₋₄alkoxy, —CN, Cl, Br, I, —NO₂, —NH₂, —NHCOR⁹, —NHCOOR⁹, —COOH, —CONHR⁹and —COOR⁹;

R³ is selected from H, C₁₋₄ alkyl, —OH, C₁₋₄ alkoxy, —CN, Cl, Br, I,—NO₂, —NH₂, —NHCOR⁹, —NHCOOR⁹, —COOH, —CONHR⁹, —COOR⁹ and COAr¹⁰;

R⁸ and, R⁹ are independently selected from C₁₋₄ alkyl, optionallysubstituted phenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl andligands;

Ar is selected from

in which B is N or CR¹⁴;

Z is O, S —CH═CH— or NH;

the or each R¹¹ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂,—NH₂, —NHR¹⁰, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵, —COOH,—CONHR¹⁶, —NHCOOR¹⁶ and COOR¹⁶;

n is an integer in the range 0 to 4;

the or each R¹⁰ is selected from C₁₋₄ alkyl, optionally substitutedphenyl, C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

R¹² is H, —COAr¹, —CONH₂, —COOH, —COR¹⁶ or —COOR¹⁶;

the or each R¹³ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂,—NH₂, —NHR¹⁰, —NR¹⁰ ₂, —N⁺R¹⁰ ₃, —CN, Cl, Br, I, —NHCOR¹⁵, —COOH,—CONHR¹⁶, —NHCOOR¹⁶ and —COOR¹⁶;

m is 0, 1 or 2;

R¹⁴ is selected from OH, C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —NH₂, —CN, Cl,Br, I, —NHCOR¹⁵, —COOH, —CONHR¹⁶, —NHCOOR¹⁶—COOR¹⁶ and H;

R¹⁵ is selected from C₁₋₄ alkyl, optionally substituted phenyl,optionally substituted heteroaryl, C₇₋₁₂ aralkyl, Ar¹ and ligands;

R¹⁶ is selected from C₁₋₄alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

Ar¹⁰ is

in which x is 0, 1 or 2;

Ar¹ is selected from the same groups as Ar; provided that no more thanone group R¹¹ or R¹³ in any one ring includes a group Ar¹.

The animal which is treated is generally a human, although the compoundsmay also have veternary use. The indication treated is generally cancer,including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The tumour may, forinstance, be defined s as a tumour expressing high levels of CYP1B1.

In the invention a group Ar¹ is preferably

In the invention, the leaving group Y is, for instance, a group whichhas utility as a leaving group in nucleophilic substitution reactions.Suitable examples of such groups are —OCOOR⁵, —OCONHR⁶, Cl, Br, I, or—OSOOR⁷, in which R⁵, R⁶ and R⁷ are independently selected fromC₁₋₄alkyl, optionally substituted phenyl, C₇₋₁₂-aralkyl and optionallysubstituted heteroaryl. Most preferably the leaving group is a halogenatom, preferably chlorine.

Optional substituents in phenyl, aralkyl and heteroaryl groups are, forinstance, C₁₋₄-alkyl, halogen, hydroxyl, C₁₋₄-alkoxy, —NH₂, —NHR¹⁰—,—NR¹⁰ ₂, —N⁺R¹⁰ ₃, —NO₂—, —CN, —COOH, —NHCOR¹⁵, —COOR¹⁶,—NHCOOR¹⁶CONHR¹⁶ etc.

In the present invention the term ligand includes a group havingspecific targeting characteristics, useful for instance in antibody orgene-directed enzyme prodrug-type environments. A ligand may be anoligopeptide, biotin, avidin or streptavidin, a polymeric group, anoligonucleotide or a protein. Preferably it has specific bindingcharacteristics such as an antibody or fragment, an antigen, a sense oranti-sense oligo-nucleotide, or one of avidin, streptavidin and biotin,that is it is one component of a specific binding pair. Alternatively itmay be a group designed for passive targeting, such as a polymericgroup, or a group designed to prolong the stability or reduceimmunogenicity such as a hydrophilic group. U.S. Pat. No. 5,843,937discloses suitable ligands for conjugating to these types of actives andmethods for carrying out the conjugation.

In a pharmaceutically active compound R¹ is other than OR⁸. In general,for optimised DNA binding ability, the group R¹ in a compound of thegeneral formula I and IA is a group Ar and/or the group R³ is a groupAr¹⁰. Often the group R¹ may include two aromatic groups joined to oneanother. In such compounds, one of the groups R¹¹ of the Ar group, orthe group R¹², as the case may be, is a group Ar¹. Whilst for somecompounds it may be desirable for three or more such aromatic groups tobe linked, it is preferred that there is one group Ar and either onegroup Ar¹⁰, or, more preferably, one group Ar¹. Thus in a group Ar¹which is a pyrrolo-dihydroindole type of group, the group R¹² should beother than a group —COAr¹. In a group Ar¹ which is one of the othertypes of group there should either be no substituents R¹¹, or R¹³ as thecase may be, or, if there are any substituents, such substituents shouldnot include a group Ar¹.

According to one embodiment of the invention, the substituent Ar is agroup

In such groups Ar, B is preferably CR¹⁴. R¹⁴ is preferably H. Thedefinition of Z is preferably NH, although furan (Z is O) and thiophene(Z is 5) analogues had been generated for conjugation to DNA alkylatingunits and may have useful DNA binding characteristics. Similarly, in agroup Ar¹, the groups B and Z are selected amongst the same preferablegroups. Preferably n is at least 1 and one of the groups R¹¹ is—NHCOAr¹. In this embodiment Ar¹ is preferably a group

in which B and Z are the same as in Ar.

In another embodiment the substituent Ar is a group

Preferably R¹² in such a group Ar is a group —COAr¹ in which Ar¹preferably is the same type of group. Alternatively R¹² in such a groupis other than —COAr¹ and R³ is —COAr¹⁰.

In both groups Ar and Ar¹, m in the indole type group is preferablyzero.

In Ar and Ar¹, there may be several substituents R¹¹. Most preferablysuch substituents are selected amongst C₁₋₄-alkoxy groups.

In compounds of the formula I, the core indole ring of the DNAalkylating sub-unit is preferably unsubstituted in the benzene ring (R²is hydrogen), whilst the pyrrole ring may be unsubstituted (R³ and R⁴are both hydrogen, or one or both of them represents a group —COOR¹⁰, ora C₁₋₄-alkyl, preferably methyl).

In the compounds of the formula I, X is H. It is believed that,hydroxylation of the compound will occur in situ at the carbon atom towhich X is attached, thereby activating the compound enabling it to actas a DNA alkylating agent.

Many of the compounds of the general formula I and IA, as well as amineprotected precursors thereof are believed to be novel compounds.According to a further aspect of the invention there is provided a newcompound of the general formula II or IIA or a salt thereof

in which R², R³ and R⁴ are as defined for formula I and IA above;

X¹ is H;

Y¹ is a leaving group;

R¹⁸ is H or an amine protecting group;

R¹⁷ is R⁸, —OR⁸—NH₂ or Ar²;

R⁸ is as defined above for formula I and IA;

Ar² is selected from

in which B¹ is N or CR⁴⁰;

R⁴⁰ is selected from H, OH, C₁₋₄-alkoxy, C₁₋₄-alkyl, —NO₂, —NH₂, —CN,Cl, Br, I, —NHCOR²², —COOH, —CONHR²³, —NHCOOR²³ and —COOR²³.

Z¹ is O, S, —CH═CH— or NR¹⁸;

the or each R¹⁹ is selected from, OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂,—NHR¹⁸, —NHR²³, —NR²³ ₂, —N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH,—CONHR²³ and —COOR²³;

p is an integer in the range 0 to 4;

R²⁰ is H, —COAr³, —CONH₂, —COOH, —COR²³ or —COOR²³;

the or each R²¹ is selected from OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂,—NHR¹⁸, —NHR²³, —NR²³ ₂, —N⁺R²³ ₃, —CN, Cl, Br, I, —NHCOR²², —COOH,—CONHR²³ and —COOR²³;

q is 0, 1 or 2

R²² is selected from C₁₋₄ alkyl, optionally substituted phenyl,optionally substituted heteroalkyl, C₇₋₁₂ aralkyl, ligands and Ar³

R²³ is selected from C₁₋₄alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl and optionally substituted heteroaryl; and

Ar³ is selected from the same groups as Ar² provided that no more thanone R¹⁹ or R²¹ in any one ring includes a group Ar³.

Ar³ is preferably.

Compounds of the formula II or IIA, in which primary or secondary aminenitrogen atoms are protected are generally deprotected before being usedin pharmaceutical compositions. Examples of amine protecting groups arebenzyl, benzyloxycarbonyl, tertiary butyloxycarbonyl (BOC),fluorenyl-N-methoxy-carbonyl (FMOC) and2-[biphenylyl-(4)]-propyl-2-oxycarbonyl. In particularly useful servicesof compounds of the general formula II and IIA R¹⁷ is —OR⁸ and R⁸ is anamine protecting group different to R⁸OCO—. In another preferredservices R¹⁷ is other than DR⁸. Where more than one such amine group isprotected in the molecule, the protecting groups may be the same ordifferent.

The present invention further provides pharmaceutical compositionscomprising compounds of the formula I or IA or salts and apharmaceutically acceptable excipient. Pharmaceutical compositions maybe suitable for intramuscular, intraperitoneal, intrapulmonary, oral or,most preferably, intravenous administration. The compositions containsuitable matrixes, for example for controlled or delayed release. Thecompositions may be in the form of solutions, solids, for instancepowders, tablets or implants, and may comprise the compound of theformula I in solid or dissolved form. The compound may be incorporatedin a particulate drug delivery system, for instance in a liquidformulation. Specific examples of suitable excipients include lactose,sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose, such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,including arabic and tragacanth; and proteins, such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, and alginicacid or a salt thereof, such as sodium alginate. Solid compositions maytake the form of powders and gels but are more conveniently of a formedtype, for example as tablets, cachets or capsules (including spansules).Alternative, more specialised types of formulation including liposomes,nanosomes and nanoparticles.

Compounds of the formula I and IA may be synthesised using techniquesanalogous to those summarised by Boger et al 1997, op. cit. It isconvenient to form the DNA alkylating sub unit in one series of stepsand to attach this through the nitrogen atom of the dihydro-pyrrole ortetrahydroquinoline, as the case may be, (C) ring to the rest of themolecule. The DNA alkylating sub-unit may be conjugated to DNA bindingsub-units synthesised as described in Boger et al, 1997 op. cit., forinstance the PDE-I and PDE-II sub-units described in that reference. TheDNA binding subunits are the groups including Ar, Ar¹ and Ar¹⁰.

According to a further aspect of the invention there is provided a newsynthetic method in which a compound of the formula IV or IVA

in which X², R² and R⁴ are as defined above;

R³⁷ is selected from the same groups as R³;

Y² is a leaving group or a hydroxyl or protected hydroxyl group; and

R²⁶ is an amine protecting group;

is reacted with a compound of the general formula VR²⁷COY³  V

in which R²⁷ is selected from C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and Ar⁴;

Ar⁴ is selected from

in which B² is N or CR³²;

Z² is O, S, —CH═CH— or NR³³;

the or each R²⁸ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl,Br, I, —NHR³³, —NR³⁶ ₂, —N+R³⁵ ₃—, —NHCOR³⁴, —COOH, —CONHR³⁵ and—COOR³⁵;

r is an integer in the range 0 to 4;

R²⁹ is an amine protecting group;

R³⁰ is an amine protecting group, —CONH₂, —COOH, —COR³⁵ or —COAr⁵;

the or each R³¹ is selected from C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl,Br, I, —NHR³³, —NR³⁶ ₂, —N⁺R³⁶ ₃—, NHCOR³⁴, —COOH, —CONHR³⁵ and —COOR³⁵;

s is 0, 1 or 2;

R³² is selected from H, C₁₋₄-alkoxy, C₁₋₄-alkyl, NO₂, CN, Cl, Br, I,NHCOR³⁴, —COOH, —CONHR^(35,)—NHCOOR³⁵ and COOR³⁵;

the or each R³³ is an amine protecting group;

R³⁴ is selected from Ar⁵, C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

R³⁵ is selected from C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and ligands;

each R³⁶ is selected from C₁₋₄-alkyl, optionally substituted phenyl,C₇₋₁₂-aralkyl, optionally substituted heteroaryl and H

Ar⁵ is selected from the same groups as Ar⁴; and

Y³ is a leaving group, provided that no more than one R²⁸ or R³¹ in anyone ring includes a group Ar⁵.

Ar⁵ is preferably

Y³ is, for instance, selected amongst the preferred leaving groupslisted above for Y. Most suitably the definition of Y³ is Cl.Alternatively, the group Y³ may be OH. In this case, it may be necessaryto include a coupling agent to assist in the coupling reaction.

The reaction between the compound of the general formula IV or IVA andthe carboxylic acid or derivative of the general formula V is carriedout under conditions allowing such coupling to take place. Suchconditions are similar to those generally used for formation of peptidebonds, for instance as used in peptide synthetic methods.

Y² is a hydroxy group or a leaving group, which may be the same as Y ormay be converted to Y in a subsequent step.

Where, in the product, R³ is a different group to R³⁷, the R³⁷ group issubsequently derivatised to generate the desired group R³. This is oftenthe case where, for instance, R³ is a group Ar¹⁰ or —CONHR⁹. To producecompounds of that type the group R³⁷ is, for instance, —COOH or —COOR¹⁰and is reacted, optionally after hydrolysis/deprotection of a group—COOR¹⁰, with an appropriate amine compound (R⁹NH₂ or Ar¹⁰H), optionallyin the presence of coupling agents, to produce the amide linkedcompound.

After the coupling process, it may be desirable to deprotect one or moreof the protected amine groups. If further reaction, for instance withother derivatising agents such as glycosyl compounds, peptides, polymersetc is desired through any such amine groups, it may be desirable todeprotect only those to which subsequent reaction to to take place,whilst retaining the other amine groups in a protected form. Selectionof suitable amine protecting groups and protection and deprotectionprotocols may be made using techniques commonly utilised in peptidechemistry.

It is believed that some of the intermediates of the general formula IVor IVA may be novel compounds. According to a further aspect of theinvention, there is provided a novel compound of the general formula IIIor IIIA

in which R² and R⁴ are as defined for formula I and IA above;

R³⁸ is selected from the same groups as R³;

X² is H;

Y² is a leaving group or a hydroxyl or protected hydroxyl group; and

R²⁴ and R²⁵ are each H or an amine protecting group.

In compounds of the general formula III and IIIA, in the compound readyfor reaction with a carboxylic acid derivative, for instance of thegeneral formula V, R²⁴ is H, whilst R²⁵ should be an amine protectinggroup. Precursors for such compounds may have both ring nitrogen atomsin protected form, that is in which R²⁵ and R²⁴ represent protectinggroups. In such compounds, since it is desired for the compound to becapable of derivatisation at just one of the nitrogen atoms, preferablyR²⁴ and R²⁵ represent different protecting groups.

In compounds of the formula III and IIIA, the group Y² may be selectedamongst those defined above for leaving group Y. The nature of the groupY² should be selected having regard to the nature of the reagent withwhich the compound of the formula IV or IVA, as the case may be, is toreact in a subsequent step. Suitable examples of leaving group Y² areselected from those listed above for Y.

The compound of the formula III may be prepared in a preliminary stepusing as the starting material an aniline compound having a leavinggroup substituent Y⁴ at the carbon atom ortho to the amine groupsubstituent, and an N-substituent which is a trans 2-propen-1-yl group—CH₂CH═CHY⁵, in which Y⁵ is hydrogen or a group which is the same as Y²or may be converted to into Y² in a subsequent step in which the anilinederivative is reacted under cyclisation conditions, to form adihydropyrrole ring. Preferably in the cyclisation reaction a halogenY⁵(═Y²) substituent is retained. The group Y⁴ should be a radicalleaving group, such as halogen, preferably I or Br. Suitable radicalsfor carrying out the cyclisation reaction where Y⁵ is hydrogen arenitroxy compounds such as 2,2,6,6-tetramethylpiperidinyloxy (TEMPO).Where Y⁵ is a radical leaving group (gY²) the reaction may be carriedout in the presence of a radical derived from azoisobutyronitrile(AIBN). In this step Y⁵ does not leave. Suitable catalysts for a radicalcyclisation step are tin hydride compounds such as tributyl tin hydride.This synthetic pathway is illustrated in Examples 1 and 3.

The compound of the general formula IIIA may be formed by cyclisation ofan aniline compound having a radical leaving group Y⁴ substituent orthoto the amine group and an N-substituent which is a 2-propen-l-yl group,preferably a trialkyl tin radical, under cyclisation conditions to forman intermediate dihydroquinonone. The cyclisation reaction is conductedin the presence of suitable catalysts which are, for instance, palladiumcomplexes such as tetrakis (triphenylphosphine) palladium (0),bis(triphenyl phosphine) palladium (II) chloride or palladium (II)acetate. The dihydroquinonine intermediate is oxidised to form a furtherintermediate which is an epoxide, for instance using a peroxide reagent.The epoxide intermediate is reduced using a suitable selective reducingagent such as a dialkyl aluminium hydride to produce the correspondingalcohol which is subsequently halogenated, for instance using carbontetrachloride/triphenyl phosphine. This reaction is illustrated inExamples 2 and 4.

The starting compound for such reactions may be represented by thegeneral formula VI

in which R², R⁴, R²⁶, and X² are the same as in the compound of theformula IV;

R³⁹ is selected from the same groups as R³;

R⁴⁰ is an amine protecting group different from R²⁶,

one of Z¹ and Z² is Y⁵ and the other is H;

Y⁵ hydrogen, or is a leaving group which is the same as or different toY²; and

Y⁴ is a radical leaving group.

Y⁴ is preferably selected from Cl, Br and I.

The compound of the general formula VI may be produced by alkylation ofthe sodium salt of the corresponding amiline derivative with a cis ortrans-1,3-dihalo prop-2-ene compound. The cis starting material producesa compound of the general formula IV in which Z² is Y⁵, the transstating material a product VI in which Z¹ is Y⁵. An allyl reagentproduces a compound VI in which Y⁵ is hydrogen.

The carboxylic acid derivative of the general formula V may besynthesised using the methods generally described in Boger et al, 1997op.cit, for instance PDE-I and PDE-II may be synthesised using theUmezawa synthesis, the Rees-Moody synthesis, the Magnus synthesis, theCava-Rawal synthesis, the Boger-Coleman synthesis, the Sundbergsynthesis, the Martin synthesis, the Tojo synthesis. Indole-2-carboxylicacid is commercially available. Other analogues of the DNA bindingsub-units of the duocarmycins, and reactive carboxylic acid derivativesthereof are described by Boger et al, op.cit. and in U.S. Pat. No.5,843,937.

Two specific examples of compounds of the general formula I and II are

Two specific examples of a compound of the general formula IA and IIAare:

Other examples are ethyl rather than methyl esters of compounds VII andX.

The present invention relates to the creation of a range of prodrugsthat have little or no cytotoxic effects when in their normal state, butare highly cytotoxic (i.e. have a substantially increased cytotoxicity)when activated by oxidation or hydroxylation by CYP enzymes. Thisprovides for a self-targeting drug delivery system in which a noncytotoxic (or negligibly cytotoxic) compound can be administered to apatient, for example in a systemic manner, the compound then beingactivated at the site of the tumour cells (intratumoural activation) toform a highly cytotoxic compound which acts to kill the tumour cells.The fact that the CYP isoforms are not expressed by normal cells meanthat the activation of the compound only occurs at the site of thetumour cells and therefore only tumour cells are affected, thusproviding a self-targeting system.

The prodrugs of the present invention have the distinct advantage ofbeing useful in the treatment of tumours at any site in the body,meaning that even tumours that have undergone metastasis (which arenormally not susceptible to site specific therapies) may be treated.

The prodrug may be an antitumour prodrug. Examples of tumours includecancers (malignant neoplasms) as well as other neoplasms e.g. innocenttumours. The prodrug may be activated by hydroxylation by isoforms ofcytochrome P450's.

In a variation of the normal procedure which relies upon CYP expressionwithin tumour cells to effect selective hydroxylation and henceactivation of the prodrugs, the selectivity between tumour tissue andnormal tissue can be enhanced in a two part procedure. Thus (a)infecting tumor cells with a viral vector carrying a cytochrome P450gene and a cytochrome P450 reductase gene, wherein expression ofcytochrome P450 gene and cytochrome P450 reductase gene by tumor cellsenables the enzymatic conversion of a chemotherapeutic agent to itscytotoxic form within the tumor, whereby the tumor cells becomeselectively sensitized to the prodrug chemotherapeutic agent (b)contacting tumor cells with the prodrug chemotherapeutic agent wherebytumor cells are selectively killed.

These prodrugs are pyrrolodihydroindole (general formula I) or pyrrolotetrahydroquinoline (general formula IA) derivatives. Their specific useas antitumour prodrugs has not been previously suggested or disclosed,nor has the suggestion that they are prodrugs having an activatedhydroxylated form. Where compounds of formula (I) have been previouslyidentified and made, they have not been identified as anti-tumour agentsdue to their poor (or negligible) cytotoxicity. Thus the intratumouralhydroxylation of the prodrugs of the present invention provides themwith a surprising and unexpected efficacy.

Hydroxylated forms of the prodrugs are potent DNA alkylating agents thatbind in the minor groove of DNA and alkylate the purine bases at the N3position. As such, they are potent cytotoxic agents whose exactbiological mechanism of action is unknown but involves the disruption oftemplate and other functions of DNA. General inhibition of templatefunction of DNA will affect and be generally cytotoxic to all dividingcells in the body and lead to unacceptable side effects in a therapeuticsetting. However, the targetted production of hydroxylated forms only intumour cells that overexpress particular isoforms of cytochrome P450'swill lead to a specific cytotoxic effect only in those cells. Thenon-hydroxylated forms are essentially non-toxic to all cells.

The following examples illustrate the invention:

EXAMPLE 1

The synthesis of one compound of the general formula I is carried outaccording to the following reaction scheme.

1.1 1-Benzoyl-5-nitroindole

5-nitroindole (100 mg, 0.62 mmol) in CH₂Cl₂ (1 mL) is treated withbenzoyl is chloride (86 mg, 0.62 mmol, 1 equiv.) and4-dimethylaminopyridine (74 mg, 0.62 mmol, 1 equiv.). The mixture isstirred at room temperature for 1 h, diluted with CH₂Cl₂ (10 mL), washedwith HCl (1M, 2×10 mL) and water (1×10 mL), dried (MgSO₄) andconcentrated. Chromatography (Silica gel, 2×15 cm, 10% ethylacetate/hexanes) gives the product.

1.2 2-Amino-1-benzoylindole

1-Benzoyl-5-nitroindole (100 mg, 0.38 mmol) in ethyl acetate (2 mL) istreated with 10% Pd/C (10 mg) and stirred under an atmosphere ofhydrogen at room temperature for 4 h. The resulting solution is filteredthrough celite and concentrated. Chromatography (Silica gel, 2×15 cm,10% ethyl acetate/hexanes) gives the product.

1.3 5-Amino-1-benzoyl-4-iodoindole

5-Amino-1-benzoylindole (100 mg, 0.42 mmol) in tetrahydrofuran (THF) (1mL) is treated with N-iodosuccinimide (103 mg, 0.46 mmol, 1.1 equiv) and4-toluenesulfonic acid (16 mg, 0.08 mmol. 0.2 equiv.) and stirred atroom temperature (RT) for 16 hours. The solution is concentrated andredissolved in ethyl acetate (10 mL). The organic layer is washed withwater (1×10 is mL), 1M HCl (2×10 mL) and water (1×10 mL), dried (MgSO₄)and concentrated. Chromatography (Silica gel, 2×15 cm, 10% ethylacetate/hexanes) gives the product.

1.4 1-Benzoyl-5-((tert-butyloxy)carbonyl)amino-4-iodoindole

5-Amino-1-benzoyl4-iodoindole (100 mg, 0.28 mmol) is stirred in CH₂Cl₂(2 mL) and treated with di-tert-butyl-dicarbonate (89 mg, 0.41 mmol, 1.5equiv), triethylamine (57 μL, 0.41 mmol, 1.5 equiv) and4-dimethylaminopyridine (4 mg, 0.028 mmol, 0.1 equiv). After 16 h at RT,the solvents are removed under reduced pressure. Chromatography (Silicagel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

1.51-Benzoyl-5-[N-(3-Chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole

1-Benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg, 0.22mmol) was stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66 mmol,60% dispersion in oil, 3 equiv.) is added. After 15 min, the suspensionis treated with E/Z-1,3-dichloropropene (61 μL, 0.66 mmol, 3 equiv) andthe resulting solution was stirred at RT for 16 h. The solution isconcentrated and water (10 mL) is added. The aqueous solution wasextracted with ethyl acetate (3×10 mL), the organic layers combined,dried and concentrated. The product was obtained after chromatography(Silica gel, 2×15 cm, 10% ethyl acetate/hexanes).

1.61-(Chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole

1-Benzoyl-5-[N-(3-Chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(100 mg, 0.19 mmol), poly(methylhydrosiloxane) (200 μL),bis(tributyltin) oxide (19 μL, 0.04 mmol, 0.2 equiv) and azoisobutyronitrile (AIBN) (6 mg, 0.04 mmol, 0.2 equiv) were stirred intoluene (2 mL) at 80° C. under N₂ for 4 h. The solvent is then removedin vacuo. Chromatography (Silica gel, 2×15 cm, 10% ethylacetate/hexanes) gives the product.

1.7 5-Methoxyindole extended agent.1-(chloromethyl)₆-benzoyl-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole

1-(Chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole(100 mg, 0.24 mmol) is treated with a solution of hydrochloric acid inethyl acetate (4M, 500 μL). After 30 min, the solvent is concentratedand dimethyl formamide (DMF) (1 mL) is added. The solution is treatedwith 1-[(3-dimethylamino)propyl]-3-ethyl carbodimide (EDC) (140 mg, 0.73mmol) and 5-methoxyindole-2-carboxylic acid (140 mg, 0.73 mmol). After16 h, the solvent is removed under reduced pressure. Chromatography(Silica gel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product. The6-benzoyl protecting group may be removed by sodium methoxide inmethanol, followed by removal of the solvent.

EXAMPLE 2

The following example illustrates the synthesis of a compound of thegeneral formula 1A in which R¹ is OR⁸ and R⁸ is tBu. It is suitable forextending by a step analogous to step 1.7 above, to form a furthercompound of the formula IA in which R¹ is Ar, and optionallysubsequently deprotected at the nitrogen atom of the indole ring.

2.11-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole

1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg, 0.22mmol) is stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66 mmol, 60%dispersion in oil, 3 equiv.) is added. After 15 min, the suspension istreated with E/Z-1-tributylstannyl-3-bromopropene (270 mg, 0.66 mmol, 3equiv) (Boger, D. L.; McKie, J. A.; Boyce, C. W. Synlett 1997, 515-516)and the resulting solution is stirred at RT for 16 h. The solution wasconcentrated and water (10 mL) is added. The aqueous solution isextracted with ethyl acetate (3×10 mL), the organic layers combined,dried and concentrated. The product was obtained after chromatography(Silica gel, 2 15 cm, 10% ethyl acetate/hexanes)

2.21,2-Dihydro-1-((tert-butyloxy)carbonyl)-5,6-(9-benzoylpyrrolo)quinoline.

1-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(100 mg, 0.12 mmol) and tetrakis(triphenylphosphine)palladium(0) (32 mg,0.2 equiv) are stirred in toluene (2 mL) at 50° C. under N₂ for 2 h. Thesolvent is then removed in vacuo. Chromatography (Silica gel, 2×15 cm,10% ethyl acetate/hexanes) gives the product.

2.33,4-Epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoylpyrrolo)quinoline.

1,2-dihydro-1-((tert-butyloxy)carbonyl)-5,6-(9-benzoylpyrrolo)quinoline.(100 mg, 0.27 mmol) and 3-chloroperoxy benzoic acid (68 mg, 0.40 mmol,1.5 equiv) were stirred in CH₂Cl₂ (2 mL) at −78° C. to −30° C. under N₂for 2 h. The solvent is then removed in vacuo. Chromatography (Silicagel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

2.44-Hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline.

3,4-epoxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoylpyrrolo)quinoline(100 mg, 0.26 mmol) was treated with disobutyl aluminium hydride(Dibal-H) (55 mg, 0.39 mmol, 1.5 equiv) in THF (2 mL),at −78° C. to −30°C. under N₂. After 1 h, the reaction is quenched by the addition ofwater (2 mL) and the resulting solution is extracted with ethyl acetate(3×10 mL), the organic layers combined, dried and concentrated. Thesolvent is removed in vacuo. Chromatography (Silica gel, 2×15 cm, 10%ethyl acetate/hexanes) gives the product.

2.54-Chloro-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline

4-hydroxy-1-((tert-butyloxy)carbonyl)-1,2,3,4-tetrahydro-5,6-(9-benzoyl)pyrroloquinoline(100 mg, 0.26 mmol) in CH₂Cl₂ (2 mL) is treated with a prepared solutionof PPh₃ (137 mg, 0.52 mmol, 2 equiv) and CCl₄ (200 mL) in CH₂Cl₂ (2 mL)at RT. After 4 h, the solvent is removed in vacuo. Chromatography(Silica gel, 2×15 cm, 10% ethyl acetate/hexanes) gives the product.

EXAMPLE 3

3.1 Ethyl 1-benzoyl-5-nitroindole-2-carboxylate (3.2)

Ethyl 5-nitroindole-2-carboxylate (3.1) (1.5 g, 6.41 mmol) in CH₂Cl₂ (30ml) was treated with benzoyl chloride (1.19 ml, 10.26 mmol), Et₃N (891μl, 6.41 mmol) and DMAP (783 mg, 6.41 mmol). The mixture was stirred for16 h. 10% NaHCO₃ (10 ml) and CH₂Cl₂ (10 ml) were added and the organiclayer was separated. The aqueous layer was extracted with CH₂Cl₂ (3×5mL). The combined organic layers were washed with H₂O (10 ml), 5% HCl(10 ml) and H₂O (10 ml). The solution was dried (MgSO₄) andconcentrated. The 10 residue was crystalised from EtOAc/Hex 1:9 toafford 1.85 g (87%) of 3.2 as a yellow powder: ¹H NMR (250 MHz, CDCl₃) δppm 8.66 (d, 1H), 8.25 (d, 1H), 7.80 (d,1H), 7.6-7.72 (m, 3H), 7.48 (m,3H), 4.00 (q, 2H), 1.10 (t, 3H). FABMS (NBA/NaI) m/z 339 (M+H⁺ expected339)

3.2 Ethyl 5-amino-1-benzoylindole-2-carboxylate (3.3).

A solution of 3.2 (1.86 g, 5.5 mmol) and 10% Pd/C (440 mg) in dry THF(30 ml) was stirred under H₂ for 16 hrs. The resulting mixture wasfiltered through celite which was washed with EtOAc (40 ml) and thefiltrate was concentrated. The residue was purified by chromatography(SiO₂, 0 to 40% EtOAc in hexanes) to afford 3.3 (1.63 g, 96%) as abright yellow oil. ¹H NMR (250 MHz, CDCl₃) δ ppm 7.40-7.72 (m, 6H), 7.18(s, 1H), 6.92 (d, 1H), 6.82 (dd, 1H), 3.92 (q, 2H), 3.68 (br s, 2H),1.06 (t, 3H); FABMS: (NBA/NaI) m/z 308 (M+H⁺ expected 308).

3.3 Ethyl 5-amino-1-benzoyl-4-iodoindole-2-carboxylate (3.4)

5-amino-1-benzoylindole (1.63 g, 5.29 mmol) in THF (75 mL) was treatedwith N-iodosuccinimide (1.89 g, 8.46 mmol) and 4-toluenesulfonic acid(364 mg, 2.12 mmol) and stirred at RT for 16 hours. The solution wasconcentrated and redissolved in ethyl acetate (100 mL). The organiclayer was washed with water (1×100 mL), 1 M HCl (2×100 mL) and water(1×100 mL), dried (MgSO₄) and concentrated. Chromatography (SiO₂, 10%ethyl acetate/hexanes) gave the product (1.17 g, 51%) as a bright yellowsolid. ¹H NMR (250 MHz, CDCl₃) δ ppm 7.30-7.70 (m, 6H), 7.30 (s, 1 H),6.80 (d, 1 H), 4.05 (s, 2 H), 3.85 (q, 2 H), 1.0 (t, 3H) FABMS (NBA/NaI)m/z 434 (M+H⁺ expected 434),457 (M+Na⁺ expected 457).

3.4 Ethyl1-benzoyl-5-(N-(tert-butyloxycarbonyl)-4-iodoindole-2-carboxylate (3.5)

A mixture of 3.4 (1.17 g, 2.70 mmol), (Boc)₂O (9.40 g, 43 mmol) and Et₃N(375 μL, 2.70 mmol) in dioxan (100 mL) was heated to 100° C. under N₂for 48 h. Upon completion, the solution was cooled, concentrated andpurified by flash chromatography (SiO₂, 0-20% EtOAc in hexane) to afford3.5 (1.3 g, 90%) as a yellow oil. FABMS (NBA.NaI) 535 (M+H⁺ expected535).

3.5 Ethyl1-benzoyl-5-[N-(3-chloro-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole(3.6)

1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (100 mg, 0.22mmol) was stirred in DMF (1 mL) and sodium hydride (26 mg, 0.66 mmol,60% dispersion in oil, 3 equiv.) was added. After 15 min, the suspensionwas treated with E/Z-1,3-dichloropropene (61 μL, 0.66 mmol, 3 equiv) andthe resulting solution was stirred at RT for 16 h. The solution wasconcentrated and water (10 mL) was added. The aqueous solution wasextracted with ethyl acetate (3′10 mL), the organic layers combined,dried and concentrated. The (3.6) product was obtained afterchromatography (SiO₂, 10% ethyl acetate/hexanes) as a yellow oil (125mg, 94%). FABMS (NBA/NaI) m/z 609 (M+H⁺ expected 609).

3.6 Ethyl6-benzoyl-1-(chloromethyl)-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.7)

Compound 3.6 (100 mg, 0.19 mmol), and AIBN (6 mg, 0.04 mmol, 0.2 equiv)were stirred in toluene (2 mL) at 80° C. under N₂. Bu₃SnH (51 μL, 0.19mmol) was added in 4 portions over 1 h. The solvent was then removed invacuo. Chromatography (SiO₂ 10% ethyl acetate/hexanes) gave the product(3.9) (72 mg, 78%) as an oil. FABMS (NaI/NBA) m/z 483 (M+H+expected483).

3.7 Ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.8)

Compound 3.7(100 mg, 0.21 mmol) was treated with a solution ofhydrochloric acid in ethyl acetate (4M, 500 μL). After 30 min, thesolvent was concentrated and DMF (1 mL) was added. The solution wastreated with EDC (120 mg, 0.63 mmol) and 5-methoxyindole-2-carboxylicacid (120 mg, 0.63 mmol). After 16 h, the solvent was removed underreduced pressure and the residue (the 6N-benzoyl protected precursor ofcompound 3.8) was dissolved in CH₃OH (1 mL). A solution of NaOCH₃ inCH₃OH (2M, 100 μL) was then added and the solution stirred for 10minutes. The solvent was removed and chromatography (SiO₂, 10% ethylacetate/hexanes) gave the product (3.8) (100 mg, 86%). FABMS (NBA/NaI)m/z 557 (M+H⁺ expected 557).

EXAMPLE 4

4.1 Ethyl1-Benzoyl-5-[N-(3-(tributylstannyl)-2-propen-1-yl)-N-((tert-butyloxy)carbonyl)]amino-4-iodoindole-7-carboxylate(4.1)

Ethyl 1-benzoyl-5-(tert-butyloxycarbonyl)amino-4-iodoindole (3.5,synthesised as described in Example 3.1-3.4) (100 mg, 0.18 mmol) wasstirred in DMF (1 mL) and sodium hydride (21 mg, 0.54 mmol, 60%dispersion in oil, 3 equiv.) was added. After 15 min, the suspension wastreated with E/Z-1-tributylstannyl-3-bromopropene (221 mg, 0.54 mmol, 3equiv) and the resulting solution was stirred at RT for 16 h. Thesolution was concentrated and water (10 mL) was added. The aqueoussolution was extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The product was obtained afterchromatography (SiO₂, 10% ethyl acetate/hexanes) as a colourless solid(132 mg, 92%). FABMS (NBA/NaI) m/z 792 (M+H⁺ expected 792).

4.25,6-(9-Benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-2,4-dihydroquinoline(4.2)

Compound 4.1 (100 mg, 0.12 mmol) andtetrakis(triphenylphosphine)palladium(0) (32 mg, 0.2 equiv) were stirredin toluene (2 mL) at 50° C. under N₂ for 2 h. The solvent was thenremoved in vacuo. Chromatography (SiO₂, 10% ethyl acetate/hexanes) gavethe product (50 mg, 94%) as a yellow oil. FABMS (NBA/NaI) m/z 447 (M+H⁺expected 447).

4.35,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-3,4-epoxy-1,2,3,4-tetrahydroquinoline(4.3)

Compound 4.2 (100 mg, 0.22 mmol) and MCPBA (57 mg, 0.33 mmol, 1.5 equiv)were stirred in CH₂Cl₂ (2 mL) at −30° C. under N₂ for 2 h. The solventwas then removed in vacuo. Chromatography (SiO₂, 10% ethylacetate/hexanes) gave the product (70 mg, 69%) as an oil. FABMS(NBA/NaI) m/z 463 (M+H⁺ expected 463).

4.45,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-4-hydroxy-1,2,3,4-tetrahydroquinoline(4.4)

Compound 4.3 (100 mg, 0.22 mmol) was treated with Dibal-H (46 mg, 0.33mmol, 1.5 equiv) in THF (2 mL), at −30° C. under N₂. After 1 h, thereaction was quenched by the addition of water (2 mL) and the resultingsolution was extracted with ethyl acetate (3×10 mL), the organic layerscombined, dried and concentrated. The solvent was removed in vacuo.Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the alcohol (85mg, 83%). FABMS (NBA/NaI) m/z 465 (M+H⁺ expected 465).

4.55,6-(9-benzoyl-8-(ethyloxy)carbonylpyrrolo)-1-((tert-butyloxy)carbonyl)-4-chloro-1,2,3,4-tetrahydroquinoline(4.5)

Compound 4.4 (100 mg, 0.22 mmol) in CH₂Cl₂ (2 mL) was treated with aprepared solution of PPh₃ (116 mg, 0.44 mmol, 2 equiv) and CCl₄ (200 μL)in CH₂Cl₂ (2 mL) at RT. After 24 h, the solvent was removed in vacuo.Chromatography (SiO₂, 10% ethyl acetate/hexanes) gave the targetcompound as an oil (95 mg, 90%). FABMS (NBA/NaI) m/z 484 (M+H⁺expected484). The compound may be deprotected by removal of the tBOC group, aDNA-binding sub-unit conjugated to the nitrogen atom of the tCtrahydroquinoline ring and the indole nitrogen subsequently deprotected bysteps analogous to those of Example 3.7.

EXAMPLE 5 Biological Testing of Ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate(3.8)

Materials and Methods

5.1 Incubation Mixtures of Compound and Microsomes

Test compound (synthesised in example 3) activation by CYP enzymes wascarried out using NADPH supplemented rat liver microsomes; Incubationmixtures comprised microsomal protein (1 mg/ml), reduced-nicotinamideadenine dinucleotide phosphate (NADPH, 10 mM) and phosphate buffer(pH7.4, 100 mM). Test compound (0.01-100 μM final concentration) in DMSO(20 μl) was added to the microsomal incubation mixtures (0.5 ml) andincubated for 60 min at 37C. Control incubates contained test compoundand microsomal incubation mixture terminated at 0 time. All incubationswere terminated by addition of an equal volume of ice-cold acetonitrileand microfuged for 3 min. Aliquots of the supernatant were added tocells in culture.

5.2 Cell Culture Based Cytotoxicity Measurement

Chinese Hamster Ovary (CHO) cell were grown in MEM supplemented with.10% dialysed FBS and G418 (400 μg/ml). All cells were seeded at aninitial density of 1000 cells/well in 96-well-plates, incubation at 37°C. for 24 hours. Aliquots (0.1 ml) of the testcompound/microsomal/acetonitrile supernatnant was then added to the CHOcells. Cells were then incubated for 24 hours at 37° C., 5% CO₂. Afterthis time period MTT (50 μl; 2 mg/ml stock solution) was added to eachwell and cells were incubated for a further 4 hours. During this timeperiod MTT, a hydrogen acceptor tetrazolium salt, is reduced to formazandye by mitochondrial dehydrogenase of viable cells. The media wasaspirated from cells and DMSO (100 μl/well) added to solubilise thecoloured formazan dye. Absorbance of the formazan dye in the96-well-plates was then determined at 550 nm. The effect of microsomalactivation by the test compound on the arrest of CHO cell growth couldbe determined by comparing the IC₅₀ (concentration that inhibited cellgrowth by 50%) with and without microsomal incubation.

Results CHO IC50 (μM) compound +activation −activation AF 3.8 0.06 ±0.02 4.3 ± 0.41 71.7*

Effect of compound 3.8 and its metabolism (activation) product on thesurvival of Chinese hamster ovary cells in culture. Cells were incubatedfor 24 hours with supernatants from reaction mixtures of compound 3.8with NADPH fortified rat liver microsomes. IC₅₀ represents theconcentration of drug required to inhibit cell growth by 50%. Values areexpressed as the mean±sd for three experiments. See methods for fulldetails of metabolism. AF=activity factor i.e. the ratio of IC₅₀cytotoxicity values obtained for ±compound 3.8 activation.

-   -   * represents significance at p>0.05.

1. A pharmaceutical composition comprising a compound of the generalformula I and a pharmaceutically acceptable excipient in which B is CH;Z is NH; the or each R¹¹ is selected from the group consisting of —OH,C₁₋₄ alkoxy, C₁₋₄ alkyl, —NO₂, —CN, Cl, Br, I, —NHCOR¹⁵, and COOR¹⁶; nis an integer in the range 0 to 4; R¹² is selected from the groupconsisting of H, —CONH₂, and —COR¹⁶; the or each R¹³ is selected fromthe group consisting of OH and C₁₋₄ alkoxy; m is 0, 1 or 2; R¹⁵ is C₁₋₄alkyl; and R¹⁶ is C₁₋₄ alkyl.
 2. A compound of the general formula II ora salt thereof

in which R² is H; R⁴ is selected from the group consisting of H, C₁₋₄alkyl, and —COOR⁹; R³ is selected from the group consisting of H, C₁₋₄alkyl, and —COOR⁹; R⁹ is C₁₋₄ alkyl; X¹ is H;

in which X is H; Y is Cl, Br, or I; R¹ is —Ar; R² is H; R⁴ is selectedfrom the group consisting of H, C₁₋₄ alkyl, Cl, Br, I, and —COOR⁹; R³ isselected from the group consisting of H, C₁₋₄ alkyl, and —COOR⁹; R⁹ isC₁₋₄ alkyl; Ar is selected from the group consisting of

Y¹ is Cl, Br, or I; R¹⁸ is H or an amine protecting group selected fromthe group consisting of benzyl, benzoyl, benzyloxycarbonyl,t-butyloxycarbonyl, fluorenyl-N-methoxycarbonyl and2-[biphenylyl-(4)]-propyl-2-oxycarbonyl; R¹⁷ is Ar²; Ar² is selectedfrom the group consisting of:

in which B¹ is CH; Z¹ is NR¹⁸; the or each R¹⁹ is selected from thegroup consisting of OH, C₁₋₄ alkoxy C₁₋₄ alkyl, NO₂, —CN, Cl, Br, I,—NHCOR²², and —COOR²³; p is an integer in the range 0 to 4; R²⁰ isselected from the group consisting of H, —CONH₂, and —COR²³; the or eachR²¹ is selected from OH and C₁₋₄ alkoxy; q is 0, 1 or 2; R²² is C₁₋₄alkyl; and R²³ is C₁₋₄ alkyl.
 3. A compound according to claim 2 inwhich the or each R¹⁸ is H.
 4. A compound according to claim 2 in whichY¹ is Cl.
 5. A compound according to claim 2 in which R³ is H or COOMe.6. A compound according to claim 2 in which R⁴ is H.
 7. A compoundselected from the group consisting of:1-(chloromethyl)-6-benzoyl-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;1-(chloromethyl)-6-benzoyl-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole;ethyl6-benzoyl-1-(chloromethyl)-3-((tert-butyloxy)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate;ethyl6-benzoyl-1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate; and ethyl1-(chloromethyl)-3-((5-methoxy-1H-indol-2-yl)carbonyl)-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate.8. A pharmaceutical composition comprising a compound according to claim2 and a pharmaceutically acceptable excipient.
 9. A compound of thegeneral formula III

in which R² is H; R⁴ is selected from the group consisting of H, C₁₋₄alkyl, and —COOR⁹; R⁹ is C₁₋₄ alkyl; R³⁸ is selected from the groupconsisting of H, C₁₋₄ alkyl, and —COOR⁹; X² is H; Y² is Cl, Br, or I;and R²⁴ and R²⁵ are each H or an amine protecting group selected fromthe group consisting of benzyl, benzoyl, benzyloxycoarbonyl,t-butyloxycarbonyl, fluorenyl-N-methoxycarbonyl and2-[biphenylyl-(4)]-propyl-2-oxycarbonyl.
 10. A compound according toclaim 9 in which R²⁴ and R²⁵ are different from one another.
 11. Acompound according to claim 10 in which R²⁴ is butyloxycarbonyl and R²⁵is —COPh.
 12. A compound according to claim 9 in which Y² is Cl.